Project Summary/Abstract. Since neurons are both post-mitotic and long-lived, maintaining the proteome is of particular importance. Not surprisingly, disturbances in protein homeostasis have been associated with numerous neurodegenerative disorders, as well as aging. In addition, neurons are extraordinarily large cells. How protein turnover, is regulated in time and space is thus an essential cell biological question with profound implications for neuronal function and disease. Endosomal pathways leading to lysosomal degradation of endocytosed receptors are widely studied in non-neuronal cells, however, there are significant gaps in our knowledge when it comes to how the expansive dendritic arbors of mammalian neurons regulate maturation from early to late endosomes/lysosomes and degradative flux. This is true for bulk as well as regulated degradation. Our data indicate that these knowledge gaps cannot be filled by extrapolating from studies in fibroblasts. Current barriers to progress include missing ?molecular handles? for studying the heterogeneity of late endosomes and lysosomes, lack of endogenous trackable cargos that can be used to follow degradation, and ignorance of the endosomal effectors which regulate endosomal flux in dendrites. The premise for this application rests on our own data showing that there is a retrograde, Rab7 dependent, endosomal maturation gradient in dendrites. In particular, our new data show: 1) Several dendritic receptors are short-lived. They enter early endosomes after endocytosis and rapidly mature to Rab7-positive late endosomes. Contrary to common belief, they are not degraded in dendrites, but instead are retrogradely transported to the soma/proximal dendrite in a Rab7-dependent manner. The same is true for the bulk cargo BSA: degradation does not take place in dendrites, but in somatic lysosomes. 2) Lysosomal proteases, such as Cathepsin B, are largely absent from the dendritic arbor, in culture and in vivo. Most LAMP1-containing compartments in distal dendrites are thus not degradative lysosomes. 3) Most Rab7-containing late endosomes in distal dendrites contain no LAMP1, and are thus different from late endosomes in fibroblasts which overwhelmingly contain both Rab7 and LAMP1. We refer to them as ?early? late endosomes to distinguish them from the conventional Rab7/LAMP1-positive late endosomes. These new discoveries raise a significant overarching question: How does the expansive dendritic arbor coordinate global and local needs for protein turnover of membrane proteins? We will test the hypothesis that efficient endosomal progression towards degradation is regulated in a spatial gradient by Rab7 [Aim 1], that the multifunctional Rab7 effector RILP spatially coordinates the recruitment of diverse endosomal machinery along dendrites to promote somatic degradation [Aim 2], and that Rab7-mediated endosome maturation is required for development of dendrites in immature neurons and for regulation of dendrites and spines in mature neurons [Aim 3]. Our long-term goal is to unravel the mechanisms for proteostasis in dendrites.